HPV16 E6/E7 Up-regulate The Expression Of Both HIF-1α And GLUT1 By Inhibition Of RRAD And Activation Of NF-κB In Lung Cancer Cells

Objective: Chronic infection of HPV16 E6/E7 is frequently associated with lung cancers,especially in non-smokers and in Asians.In our previous studies,we found that HPV16E6/E7 up-regulated HIF-1α at protein level and further up-regulated GLUT1 at both protein and mRNA levels in well-established lung cancer cell lines.However,there are multiple pathways involved in HPV16 E6/E7 regulation of HIF-1α expression,which are not fully understood.Chlon et al showed that E6 proteins inhibited cell apoptosis mainly by degrading p53 gene.While Todorovic et al demonstrated that E7 proteins promoted cell proliferation mainly by inhibiting retinoblastoma protein(pRb).It has been reported that RRAD is a direct target gene of p53.Therefore,we hypothesized that E6 protein inactivated RRAD by degrading p53 gene.However,the relationship between E7 protein and RRAD has not been reported.RRAD belonged to the Ras-related small GTase family,which was initially identified as a gene associated with type II diabetes and overexpressed in some patients with type II diabetes.The expression level of RRAD protein was decreased in cancer cells with poor prognosis.Zhang C et al reported that the ectopic expression of RRAD down-regulated glycolysis.Liu J et al proposed that RRAD negatively regulated the activation of NF-κB.However,the molecular mechanism of RRAD in HPV associated lung cancer is not clear.Therefore,further understanding of the regulatory mechanism of RRAD in HPV associated lung cancer will provide a new research direction for tumor research,including tumor markers and tumor targeted therapy.Methods: First,we used the technology of transfection and interference to regulate HPV16 E6 and E7,Western blotting and qRT-PCR were used to detect the expression of RRAD,p65,HIF-1α,and GLUT1 at protein and mRNA leves,the expression of p-p65 at protein level only;then we used interference technology to regulate RRAD,the expression of p65,p-p65,HIF-1α,and GLUT1 were detected by Western blotting and qRT-PCR.We used the technology of interference to regulate p65,the protein and mRNA expression of HIF-1α and GLUT1 were detected by Western blotting and quantitative real-time PCR.The expression of RRAD in lung cancer cells was regulatedby interference technology,nuclear protein and cytoplasmic protein were separated by nucleoplasm separation technology,then the expression of p65 and p-p65 in nucleus and cytoplasm were detected by Western bloting experiment.The effect of RRAD protein on the nuclear and plasma distribution of p65 protein was detected by immunofluorescence.The experimental data were processed and analyzed by SPSS22.0 statistical analysis software,which showed significant difference in P < 0.05.Results:1.The screening of lung cancer cell lines.First,based on our previous results,H460 cell line was low E6 and E7 expression cell line,and A549 and LK2 cells were high E6 and E7 expression cell lines.Therefore,we selected NCI-H460 cell line to transfect E6 and E7 expression plasmids,while in A549 and LK2 cell lines,SiRNA was used to interfere with the expression of endogenous E6 and E7.Secondly,the expression of RRAD and p65 in four lung cancer cell lines(NCI-H460,A549,LK2,H1299)were screened.High expression of RRAD was found in H460,whereas high expression of p65 was observed in A549 and LK2 cell lines.Further assays were designed and performed based on these results.We selected NCI-H460 cell line to interfere with the expression of its endogenous RRAD by SiRNA,and A549 and LK2 cell lines to interfere with the expression of its endogenous p65 by SiRNA.2.E6 and E7 downregulated the expression of RRAD and upregulated the expression of p65,p-p65,HIF-1α and GLUT1.The pEGFP-N1-E6 or E7 vectors were transiently transfected into the low expression H460 cell line,and the E6 or E7 empty vectors and mock transfections served as controls.The results showed that the overexpression of E6 or E7 significantly downregulated the expression of RRAD at both protein and at mRNA levels,but upregulated the expression of GLUT1 at both levels.The overexpression of E6 or E7 also upregulated the expression of p65,p-p65 and HIF-1α but at protein level only.3.Knocked down E6 and E7 upregulated the expression of RRAD and downregulated the expression of p65,p-p65,HIF-1α and GLUT1.To further verify the regulatory roles of both E6 and E7 on RRAD,p65,p-p65,HIF-1α,and GLUT1,we applied E6 or E7-specific siRNA to knockdown the expression of E6 or E7 in the A549 and LK2 cell lines.E6 or E7-nonspecific siRNA and mock specific siRNA were used to serve as thecontrols.The results indicated that the inhibition of both E6 and E7 upregulated the expression of RRAD at both protein and at mRNA levels,but downregulated the expression of GLUT1 at both levels.The inhibition of E6 or E7 also downregulated the expression of p65,p-p65 and HIF-1α but at protein level only.4.Knocked down RRAD up-regulation the expression of p65,p-p65,HIF-1α and GLUT1.High RRAD expression cell line H460 was selected and RRAD-specific siRNA to knockdown the expression of RRAD was performed.RRAD-nonspecific si RNA and mock specific siRNA were used to serve as the controls.The results showed that knocked down RRAD significantly upregulated the expression of p65 and p-p65 at the protein level only;and upregulated the expression of HIF-1α and GLUT1 at protein and mRNA levels.RRAD-nonspecific siRNA and mock specific si RNA showed minimal or no change.5.Knocked p65 downregulated the expression of HIF-1α and GLUT1.p65-specific siRNA to knockdown the expression of p65 in the A549 and LK2 cell lines was performed.p65-nonspecific siRNA and mock specific siRNA were used to serve as the controls.The results indicated that the inhibition of p65 downregulated the expression of HIF-1α and GLUT1 at both protein and mRNA levels.p65-nonspecific siRNA and mock specific siRNA showed minimal or no change.6.Knocked RRAD promoted p65 nuclear translocation.To further verify the regulatory role of RRAD on p65,we knocked RRAD in H460 cells.Our results showed that the inhibition of RRAD promoted p65 nuclear translocation significantly.The assays were performed by immunofluorescence techniques.7.Knocked RRAD up-regulated the expression levels of p65 and p-p65 in nuclear,but down-regulated the expression level of cytoplasmic p65.We knocked RRAD in H460 cells and we separated the proteins in the nucleus from the cytoplasm by using nuclear plasma separation technology.We found that the expression levels of p65 and p-p65 in the nuclear were up-regulated,but the expression level of cytoplasmic p65 was down-regulated.The expression level of cytoplasmic p-p65 was almost no change.Conclusion:1.HPV16 E6 and E7 proteins downregulated the expression of RRAD at both protein and at mRNA levels in lung cancer cell lines.To our knowledge this is the first studyfound that overexpression of E7 protein down-regulated RRAD.However,the mechanism of E6 and E7 proteins regulating of RRAD may be different.2.There are two theoretical reports on the transcription activity of NF-κB,one is the nuclear translocation of p65,the other is the expression of p-p65.In this study,we found that knocked RRAD activated NF-κB by promoting the nuclear translocation of p65 and increasing the expression level of p-p65 in nuclear.3.Knocked p65 downregulated the expression of HIF-1α at both protein and mRNA levels,and then downregulated the expression of GLUT1 at protein and mRNA levels in lung cancer cell lines.This result is not completely consistent with our previous results of the regulation of HIF-1α by E6 and E7 proteins,which may be related to the different regulation mechanism.